Spectroscopic Investigation of the Kinetic Mechanism Involved in the Association of Potyviral VPg with the Host Plant Translation Initiation Factor eIF4E

Figures

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  Figure 1. (A) Competition test of a mixture of eIF4E [∆1–46] and eIF4E[∆1–46/∆216–230] for m7GTP cap analog (A) and VPg (B) binding. (A) A mixture of both species was loaded on an m7GTP affinity column. The fraction eluted in 0.5 M KCl was analyzed by SDS-Page and Western blot. (B) A mixture of both species was incubated with (His)6-VPg and a Ni2+ IMAC was performed. The eIF4E [∆1–46/∆216–230] species was not retained (flow through). The eIF4E [∆1–46] species co-eluted with (His)6-VPg. Elution (250 mM imidazole). Fractions were analyzed by SDS-Page (Co) and Western blot Wt). Western blot revelation: lettuce eIF4E polyclonal antibodies. Subsequent analysis confirmed the identity of the two eIF4E species. The bands were cut from the gel and analyzed by mass spectrometry. Alternatively, they were transferred on a PVDF membrane and submitted to N-terminal sequencing. Both forms displayed the same N-terminal amino acid sequence.

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  Figure 2. Models of interaction between the LMV VPg central domain (blue) and lettuce eIF4E (mo10 isoform). (A–C), eIF4E interaction with VPg88–111, VPg88–120 and VPg107–120, respectively. The coordinates of the LMV VPg (LMV-AF199 isolate GenBank: AJ278854.1) and lettuce eIF4E (mo10 isoform, GenBank: AF530162.1) were first derived by homology modeling from the VPg (PDB 6NFW) and eIF4E (pisum sativum, PDB 2WMC), respectively. The binary Complex was modeled by restraint-driven docking (Haddock web server), using a set of amino acids homologous to the ones identified elsewhere [21]. V and E subscripts refer to the residues belonging to VPg and eIF4E, respectively.

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  Figure 3. Stopped-flow traces of VPg central region association with eIF4E. (A) The quenching of eIF4E tryptophan fluorescence was monitored after mixing 0.3 µM eIF4E with 1 µM VPg88–111 (final concentration). The excitation wavelength was 283 nm. Inset, plots of 1/kobs versus 1/[peptide] at 2.5, 5, 7.5 and 10 µM peptide concentration. VPg88–111 open circles, VPg88–120, open triangles. (B) Stopped-flow traces of VPg88–111*–eIF4E complex formation acquired at 520 nm upon excitation at 480 nm. VPg88–111*: doubly labeled fluorescent derivative of VPg88–111.

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  Figure 4. VPg88–111 propensity to fold into an α-helical conformation. (A) Far UV-CD spectra of VPg88–111* in the absence (dashed line), or in the presence of 10% (dotted line) and 20% (solid line) of TFE. Inset, AEDANS to fluorescein RET increase upon TFE addition (mean of three to six measurements). (B) Steady state titration of eIF4E binding to VPg88–111*. The solution contained 250 nM of VPg88–111*. AEDANS was excited at 337 nm. The solid line represents the best fit of the simple bimolecular equilibrium model to the experimental data.

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  Figure 5. Fluorescence characteristics of labeled VPg88–111. Emission spectra of 0.5 µM monolabeled AEDANS-VPg88–111 (dotted line), and dual-labeled VPg88–111* either alone (solid line) or after mixing with 1 µM eIF4E (dashed line). Excitation was 340 nm.

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  Figure 6. (A) Alignment between VPg from PVY, N strain, UniProt, P18247, used to solve the VPg structure and VPg from LMV-AF199, E strain, UniProt P89876, used to build the homology model in this study. (B) Alignment between eIF4E, Homo sapiens, UniProt P06730 and lettuce eIF4E (Salinas cultivar) UniProt Q7XJB1_LACSA.